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The synthesis of two phosphocitrate analogues and their effectiveness as calcification inhibitors


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Brown, Malcolm R(Malcolm Roy) 1984 , 'The synthesis of two phosphocitrate analogues and their effectiveness as calcification inhibitors', PhD thesis, University of Tasmania.

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The synthesis of two structural analogues of the naturally
occurring compound phosphocitrate (PC) was investigated, with a view to
producing compounds more enzymatically stable than PC, yet with similar
anti-calcifying properties. It was envisaged that such compounds might
prove to be suitable agents for preventing the deposition of insoluble
calcium salts associated with pathological calcification, especially kidney
The analogues sought were the sulphamate and phosphoramidate
derivatives of PC; namely N-phospho-2-amino tricarballylate (PAT) and
N-sulpho-2-amino tricarballylate (SAT). The preparation of PAT, which was
ultimately achieved after investigating a number of unsuccessful synthetic
routes, involved two distinct stages: a) synthesis of a new precursor in
trimethyl 2-amino tricarballylate, and b) coupling of the latter compound
with 2-cyanoethyl phosphate, followed by alkaline hydrolysis. The
preparation of SAT was effected by the coupling of 2-amino tricarballylate
with pyridine-sulphur trioxide. The same synthetic route was also utilized
to yield [ 35Si-labelled SAT.
Systems which were developed to aid in the ultimate purification
and characterization of synthetic products included ion-exchange and thin
layer chromatography, electrophoresis, isotachophoresis and chemical
assays. Additional structural proof of the new compounds was obtained
through [ 1 H]-NMR and infra-red spectroscopy.
The ability of the PC analogues to inhibit calcification in vitro
was assessed. PAT was as potent as PC in preventing hydroxyapatite
formation, while SAT was less potent but still a strong inhibitor. Both
compounds also inhibited calcium oxalate crystallization, the order of
potency being PC > SAT >> PAT.
The chemical and biological stabilities of the molecules were
studied. In vitro, PAT was found to be readily hydrolyzed at acid pH,
unstable at neutral pH and susceptible to the action of alkaline
phosphatase. SAT was shown to be completely stable at neutral and alkaline
pH, relatively stable in acid and totally resistant to the actions of the
hydrolytic enzymes sulphamatase and sulphatase. The stability of SAT was
confirmed in vivo from metabolic studies utilizing [ 35 S]-SAT. When given
orally, SAT was well absorbed across the gut and rapidly cleared unchanged
to the urine from blood and all tissues. The effectiveness of the compounds in arresting renal calcification
in vivo was also studied using well established test systems. Comparisons
were made with other inhibitors including PC. SAT was proven capable of
inhibiting calcium oxalate crystallization, whereas PC and PAT were
ineffective. With hydroxyapatite formation, different trends were observed;
PAT and SAT had no effect whereas PC produced pronounced inhibition.
Rationalization of these findings have been given.
Results presented suggest that, in terms of future possible
therapeutic value, PAT would be unsuitable, whereas PC and SAT might prove
useful under certain situations. The studies help define further the
structure-activity relationships of PC and its new analogues in terms of
anti-calcifying potential and stability. On this basis, avenues for future
research are discussed for the development of further inhibitor molecules
that might have greater activity and hence ultimately prove more useful
agents for stone prevention.

Item Type: Thesis - PhD
Authors/Creators:Brown, Malcolm R(Malcolm Roy)
Keywords: Kidneys, Urinary organs, Phosphonates, Calcification
Copyright Holders: The Author
Copyright Information:

Copyright 1984 the Author - The University is continuing to endeavour to trace the copyright
owner(s) and in the meantime this item has been reproduced here in good faith. We
would be pleased to hear from the copyright owner(s).

Additional Information:

Thesis (Ph.D.)--University of Tasmania, 1985. Bibliography: p. 152-165

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